Porphyromonas gingivalis, an anaerobic, Gram-negative bacterium, is an important organism in the pathogenesis of periodontitis. Antibiotics are used in the treatment of severe periodontitis and recently growing resistance to antibiotics in clinical P.gingivalis isolates has been reported. Metronidazole is a nitroimidazole with activity against many anaerobic bacteria, including P.gingivalis. Resistance to metronidazole has now been reported as high as 22%, but little is known about the mechanisms of resistance. In this proposal, I will use transposon mutagenesis to identify genes and gene networks involved in the development of metronidazole resistance. In my preliminary work, I have created two transposon mutant libraries in different strains of P.gingivalis using a Mariner-based transposon system. This transposon system has advantages compared to prior transposon libraries in P.gingivalis due to ability of Mariner to insert randomly as to create highly saturated libraries with insertions throughout the genome. Using this library, I have already identified previously unrecognized pigmentation mutants of P.gingivalis. Interestingly, some of these pigmentation-defective Pg mutants are resistant to metronidazole. In this proposal, I will first establish the rle of pigmentation in resistance to metronidazole by performing complementation and characterization of the pigmentation mutants I have isolated. The transposon I used is adapted for use with massively-parallel sequencing to identify the relative fitness of mutants in the libray with a technique called Tn-seq. To uncover additional non-pigment related genes involved in metronidazole resistance, I will perform a screen of the entire library using Tn-seq to identify mutants that are either enriched or eliminated on exposure to different concentrations of metronidazole. Finally, I will perform clinical sampling for P.gingivalis from patients with periodontal disease and employ targeted gene sequencing of metronidazole resistance genes identified in Aims 1 and 2, to determine whether mutations associated with in vitro resistance to metronidazole are found in patients with clinically resistant isolates of P.gingivalis. This projec is expected to shed new light on mechanisms of P.gingivalis resistance to an important antimicrobial agent. In the process I will be developing new tools and learning techniques that have the potential to be applied to multiple aspects of P.gingivalis virulence and that will prepar me for a future in oral microbiological research.